2-methylene-20(21)-dehydro-19-nor-vitamin D analogs

ABSTRACT

This invention discloses 2-methylene-20(21)-dehydro-19-nor-vitamin D analogs, and specifically 2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D 3 , and pharmaceutical uses therefor. This compound exhibits relatively high transcription activity as well as pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as an anti-cancer agent and for the treatment of skin diseases such as psoriasis as well as skin conditions such as wrinkles, slack skin, dry skin and insufficient sebum secretion. This compound also shows lower activity in vivo on bone calcium mobilization and similar in vivo intestinal calcium transport activity compared to the native hormone 1α,25-dihydroxyvitamin D 3 , and therefore may be used to treat autoimmune disorders or inflammatory diseases in humans as well as renal osteodystrophy. This compound may also be used for the treatment or prevention of obesity.

BACKGROUND OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to2-methylene-20(21)-dehydro-19-nor-vitamin D analogs and theirpharmaceutical uses.

The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog inergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity may be useful in thetreatment of a variety of diseases such as renal osteodystrophy, vitaminD-resistant rickets, osteoporosis, psoriasis, and certain malignancies.

Another class of vitamin D analogs, i.e. the so called 19-nor-vitamin Dcompounds, is characterized by the replacement of the A-ring exocyclicmethylene group (carbon 19), typical of the vitamin D system, by twohydrogen atoms. Biological testing of such 19-nor-analogs (e.g.,1α,25-dihydroxy-19-nor-vitamin D₃) revealed a selective activity profilewith high potency in inducing cellular differentiation, and very lowcalcium mobilizing activity. Thus, these compounds are potentiallyuseful as therapeutic agents for the treatment of malignancies, or thetreatment of various skin disorders. Two different methods of synthesisof such 19-nor-vitamin D analogs have been described (Perlman et al.,Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32,7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkylgroups) A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also beenprepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111(1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993); Posner etal., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)).

2-substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃ have alsobeen synthesized, i.e. compounds substituted at 2-position with hydroxyor alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with 2-alkylgroups (DeLuca et al U.S. Pat. No. 5,945,410), and with 2-alkylidenegroups (DeLuca et al U.S. Pat. No. 5,843,928), which exhibit interestingand selective activity profiles. All these studies indicate that bindingsites in vitamin D receptors can accommodate different substituents atC-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs which are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.No. 6,566,352 while 1α-hydroxy-2-methylene-19-nor-homopregnacalciferolis described in U.S. Pat. No. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. No. 6,627,622. All three of these compounds have relativelyhigh binding activity to vitamin D receptors and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α,25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents

17-ene vitamin D compounds as well as vitamin D compounds having adouble bond in the side chain thereof are also known, and have beenproposed for various pharmacological uses. Bone diseases such asosteoporosis, skin disorders such as psoriasis, cancers such as leukemiaand cosmetic conditions such as wrinkles are just some of theapplications proposed for such compounds. 17-ene compounds are describedin U.S. Pat. Nos. 5,545,633; 5,929,056 and 6,399,797 while 2-alkylidenecompounds having a side chain with a double bond therein are describedin, for example, U.S. Pat. No. 5,843,928.

SUMMARY OF THE INVENTION

The present invention is directed toward2-methylene-20(21)-dehydro-19-nor-vitamin D analogs, their biologicalactivity, and various pharmaceutical uses for these compounds. These newvitamin D compounds not known heretofore are the 19-nor-vitamin Danalogs having a methylene group at the 2-position (C-2), and a doublebond located between carbon atoms 20 and 21 in the side chain. Thepreferred vitamin D analog is2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃ (hereinafterreferred to as “Vit III 20-21”).

Structurally these 2-methylene-20(21)-dehydro-19-nor-vitamin D analogsare characterized by the general formula I shown below:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group. The preferredanalog is 2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃which has the following formula Ia:

The above compounds I, particularly Ia, exhibit a desired, and highlyadvantageous, pattern of biological activity. These compounds arecharacterized by relatively high binding to vitamin D receptors, whichis about equal to that of the native hormone 1α,25-dihydroxyvitamin D₃.These compounds also have the ability to promote intestinal calciumtransport in vivo, in a dose dependent manner, and they would beclassified as having about the same or equal intestinal calciumtransport activity, as compared to that of 1α,25-dihydroxyvitamin D₃.These compounds I, and particularly Ia, also have some ability tomobilize calcium from bone, and they would be classified as having lowerbone calcium mobilizing activity as compared to 1α,25-dihydroxyvitaminD₃. It is undesirable to raise serum calcium to supraphysiologic levelswhen suppressing the preproparathyroid hormone gene (Darwish & DeLuca,Arch. Biochem. Biophys. 365, 123-130, 1999) and parathyroid glandproliferation. These analogs having relatively low bone calciummobilization activity while very active on cell differentiation areexpected to be useful as a therapy for suppression of secondaryhyperparathyroidism of renal osteodystrophy.

The compounds I, particularly Ia, of the invention have also beendiscovered to be especially suited for treatment and prophylaxis ofhuman disorders which are characterized by an imbalance in the immunesystem, e.g. in autoimmune diseases, including multiple sclerosis,lupus, diabetes mellitus, host versus graft rejection, and rejection oforgan transplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compounds of the invention.

The above compounds I, and particularly Ia, are also characterized byrelatively high cell differentiation activity and in promotingtranscription. Thus, these compounds also provide a therapeutic agentfor the treatment of psoriasis, or as an anti-cancer agent, especiallyagainst leukemia, colon cancer, breast cancer, skin cancer and prostatecancer. In addition, due to their relatively high cell differentiationactivity, these compounds provide a therapeutic agent for the treatmentof various skin conditions including wrinkles, lack of adequate dermalhydration, i.e. dry skin, lack of adequate skin firmness, i.e. slackskin, and insufficient sebum secretion. Use of these compounds thus notonly results in moisturizing of skin but also improves the barrierfunction of skin.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of one or more of the compounds or thepharmaceutical compositions to the subject inhibits adipocytedifferentiation, inhibits gene transcription, and/or reduces body fat inthe animal subject.

One or more of the compounds may be present in a composition to treatthe above-noted diseases and disorders in an amount from about 0.01μg/gm to about 1000 μg/gm of the composition, preferably from about 0.1μg/gm to about 500 μg/gm of the composition, and may be administeredtopically, transdermally, orally, rectally, nasally, sublingually orparenterally in dosages of from about 0.01 μg/day to about 1000 μg/day,preferably from about 0.1 μg/day to about 50 μg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIGS. 1-5 illustrate various biological activities of 2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃, hereinafter referred to as“Vit III 20-21,” as compared to the native hormone1α,25-dihydroxyvitamin D₃, hereinafter “1,25(OH)₂D₃.”

FIG. 1 is a graph illustrating the relative activity of Vit III 20-21and 1,25(OH)₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor;

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of Vit III 20-21 and 1,25(OH)₂D₃;

FIG. 3 is a graph illustrating the in vitro transcription activity of1,25(OH)₂D₃ as compared to Vit III 20-21;

FIG. 4 is a graph illustrating the bone calcium mobilization activity of1,25(OH)₂D₃ as compared to Vit III 20-21; and

FIG. 5 is a graph illustrating the intestinal calcium transport activityof 1,25(OH)₂D₃ as compared to Vit III 20-21.

DETAILED DESCRIPTION OF THE INVENTION

2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃ (referred toherein as “Vit III 20-21 ”) a 19-nor vitamin D analog which ischaracterized by the presence of a methylene substituent at the carbon 2(C-2), and a double bond located between carbon atom positions 20 and 22in the side chain, was synthesized and tested. Such vitamin D analogseemed an interesting target because the relatively small methylenegroup at the C-2 position should not interfere with binding to thevitamin D receptor. Structurally, this 19-nor analog is characterized bythe general formula Ia previously illustrated herein, and its pro-drug(in protected hydroxy form) is characterized by general formula Ipreviously illustrated herein.

The preparation of 2-methylene-20(21)-dehydro-19-nor-vitamin D analogshaving the structure I can be accomplished by a common general method,i.e. the condensation of a bicyclic Windaus-Grundmann type ketone IIwith the allylic phosphine oxide III to the corresponding2-methylene-19-nor -vitamin D analog IV followed by deprotection at C-1and C-3 in the latter compound (see Scheme IV herein):

In the structures II, III and IV, groups X₁, X₂ and X₃ arehydroxy-protecting groups, preferably t-butyldimethylsilyl, it beingalso understood that any functionalities that might be sensitive, orthat interfere with the condensation reaction, be suitably protected asis well-known in the art. The process shown above represents anapplication of the convergent synthesis concept, which has been appliedeffectively for the preparation of vitamin D compounds [e.g. Lythgoe etal., J. Chem. Soc. Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev.9, 449 (1983); Toh et al., J. Org. Chem. 48, 1414 (1983); Baggiolini etal., J. Org. Chem. 51, 3098 (1986); Sardina et al., J. Org. Chem. 51,1264 (1986); J. Org. Chem. 51, 1269 (1986); DeLuca et al., U.S. Pat. No.5,086,191; DeLuca et al., U.S. Pat. No. 5,536,713].

The hydrindanone of the general structure II is not known. It can beprepared by the method shown in Schemes I, II and III herein (see thepreparation of compound Vit III 20-21).

For the preparation of the required phosphine oxides of generalstructure III, a synthetic route has been developed starting from amethyl quinicate derivative which is easily obtained from commercial(1R,3R,4S,5R)-(−)-quinic acid as described by Perlman et al.,Tetrahedron Lett. 32, 7663 (1991) and DeLuca et al., U.S. Pat. No.5,086,191.

The overall process of the synthesis of compounds I and Ia isillustrated and described more completely in U.S. Pat. No. 5,843,928entitled “2-Alkylidene-19-Nor-Vitamin D Compounds” the specification ofwhich is specifically incorporated herein by reference.

As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.Alkoxyalkyl protecting groups are groupings such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl andtetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl,diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl andanalogous alkylated silyl radicals. The term “aryl” specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,“deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively.

More specifically, reference should be made to the followingillustrative example and description as well as to Schemes I, II, IIIand IV herein for a detailed illustration of the preparation of compoundVit III 20-21.

In this example specific products identified by Arabic numerals (1, 2,3) refer to the specific structures so identified in the Schemes I, II,III and IV.

EXAMPLE

Chemistry. Ultraviolet (UV) absorption spectra were recorded with aHitachi Model 60-100 UV-vis spectrometer in the solvent noted. ¹Hnuclear magnetic resonance (NMR) spectra were recorded at 500 MHz with aBruker AM-500 FT spectrometer in deuteriochloroform. Chemical shifts (δ)are reported downfield from internal Me₄Si (δ 0.00). Mass spectra wererecorded at 70 eV on a Kratos DS-50 TC instrument equipped with a KratosMS-55 data system. Samples were introduced into the ion sourcemaintained at 120-250° C. via a direct insertion probe. High-performanceliquid chromatography (HPLC) was performed on a Waters Associates liquidchromatograph equipped with a Model 6000A solvent delivery system, aModel 6 UK Universal injector, a Model 486 tunable absorbance detector,and a differential R 401 refractometer.

Example 1

Des-A,B-23,24-dinorcholan-8β,22-diol (2). A flame dried 1000 mL twonecked flask was charged with ergocalciferol 1 (5 g, 12.6 mmol),pyridine (5 mL), and anhydrous MeOH (400 mL). The solution was cooled to−78 ° C. in an argon atmosphere. O₃ was bubbled through the solutionuntil a deep blue colour developed and persisted (about 1h). Thesolution was treated with O₂ until the blue colour faded (15 min). ThenNaBH₄ (1.5 g, 39.7 mmol) was added. After 15 min. second portion ofNaBH₄ (1.5 g, 39.7 mmol) was added and the reaction was allowed to warmto rt. Then the third portion of NaBH₄ (1.5 g, 39.7 mmol) was added andreaction stirred for over night. The reaction was quenched by addingwater (50 mL). Methanol was evaporated in vaccuo and residue wasdissolved in ethyl acetate. The organic phase was washed with 1N aqueoussolution of HCl (100 mL), saturated NaHCO₃ solution (100 mL) and brine(100 mL). The organic phase was dried (Na₂SO₄), filtered and evaporated.Purification by silica gel chromatography (25% ethyl acetate/hexane)afforded 2.18 g (10.3 mmol, 81%) of diol 2 as a white solid. Mp 110-111°C.;¹H NMR (400 MHz, CDCl₃) δ: 0.96 (3H, s), 1.03 (3H, d, J=6.6 Hz), 3.38(1H, dd, J=10.5, 6.7Hz), 3.64 (1H, dd, J=10.5, 3.2 Hz), 4.09 (1H, m);¹³C NMR (100 MHz, CDCl₃) δ: 69.2, 67.8, 52.9, 52.4, 41.8, 40.2, 38.2,33.6, 26.6, 22.6, 17.4, 16.6, 13.6; MS m/z (relative intensity): 212(M⁺, 2), 194 (M⁺−H₂O, 15), 179 (M⁺−H₂O—CH₃, 18), 125 (43), 111 (100);exact mass calculated for C₁₃H₂₂O [M−H₂O]⁺ is 194.1671, measured is194.1665.

Des-A,B-22-(p-toluenesulfonyloxy)-23,24-dinorcholan-8β-ol (3). Asolution of diol 2 (1 g, 4.71 mmol) in anhydrous pyridine (12 mL) wascooled to −25° C. and a precooled solution of tosyl chloride (1.08 g,5.66 mmol) in anhydrous pyridine (2mL) was added dropwise. The reactionmixture was stirred at that temperature for 4 h and allowed to warm to0° C. and stirred at that temperature for additional 20 h. The mixturewas diluted with CH₂Cl₂ (50 mL) and washed with saturated CuSO₄ solution(30 mL), 1N HCl (30 mL), and water (50 mL). The organic phase was dried(NaSO₄), filtered and concentrated. Purification by silica gelchromatography (25% ethyl acetate/hexane) yielded 1.7 g (4.64 mmol, 98%)of hydroxyl tosylate 3. ¹H NMR (400 MHz, CDCl₃) δ: 0.89 (3H, s), 0.96(3H, d, J=6.6 Hz), 2.45 (3H, s), 3.8 (1H, dd, J=9.2, 6.2 Hz), 3.95 (1H,dd, J=9.2, 3.0 Hz), 4.06 (1H, m), 7.35 (2H, d, J=8.2 Hz), 7.78 (2H, d,J=8.2 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 144.7, 133.0, 129.8, 127.9, 75.6,69.0, 60.4, 52.2, 41.9, 40.1, 35.7, 33.5, 26.4, 22.4, 21.6, 17.3, 16.7,13.4; MS m/z (relative integration): 366 (M⁺, 6), 194(14), 179(16),125(30), 111(100); exact mass calculated for C₂₀H₃₀SO₄Na (M+Na⁺) is389.1763, measured is 389.1768.

Des-A,B-8β-[(tert-butyldimethylsilyl)oxy]-22-(p-toluenesulfonyloxy)-23,24-dinorcholane(4). To a 0° C. cooled solution of hydroxyl tosylate 3 (1.5 g, 4.09mmol) in anhydrous DMF (20 mL) was added 2,6-lutidine (0.580 mL, 0.52 g,4.92 mmol) followed by TBSOTf (1.1 mL, 1.30 g, 4.92 mmol). The solutionwas stirred at 0° C. for 15 min and water (10 mL) was added. The mixturewas extracted with CH₂Cl₂ (3×40 mL), and combined organic phases werewashed with 1N aqueous solution of NaOH (40 mL) dried (Na₂SO₄), filteredand concentrated. The residue was purified by silica gel columnchromatography (5% ethyl acetate/hexane) to give 1.94 g (4.04 mmol, 99%)of 4. ¹H NMR (400 MHz, CDCl₃) δ: 0.01 (6H, s), 0.88 (12H, s), 0.96 (3H,d, J=6.8 Hz), 2.45 (3H, s), 3.81 (1H, dd, J=9.2, 6.4 Hz), 3.97 (1H, dd,J=9.7, 3.0 Hz), 3.99 (1H, m), 7.34 (2H, d, J=8.08 Hz), 7.79 (2H, d,J=8.2 Hz). ¹³C NMR (100 MHz, CDCl₃) δ: 114.5, 133.4, 129.8, 127.9, 74.8,69.3, 52.3, 52.6, 42.2, 40.5, 35.8, 34.4, 26.6, 25.9, 23.0, 21.6, 18.0,17.6, 16.8, 13.7, −4.8, −5.1.

Des-A,B-8β-[(tert-butyldimethylsilyl)oxy]-23,24-dinorcholan-22-al (5). Asolution of 4 (1.9 g, 3.96 mmol) in DMSO (5 mL) was added to asuspension of NaHCO₃ (1.5 g, 17.9 mmol) in DMSO (20 mL) at rt. Themixture was heated to 150° C. under argon for 15 min and cooled to rt.Water (50 mL) followed by ethyl acetate (50 mL) were added and aqueousphase was extracted with ethyl acetate (3×30 mL). The combined organicphases were dried (Na₂SO₄), filtered and concentrated. The residue waspurified by column chromatography (2% ethyl acetate/hexane) to afford0.93 g (2.87 mmol, 73%) of aldehyde 5. H¹ NMR (400 MHz, CDCl₃) δ: 0.01(6H, 2s), 0.89 (9H, s), 0.97 (3H, s), 1.09 (3H, d, J=6.8 Hz), 2.35 (1H,m), 4.03 (1H, m), 9.58 (1H, d, J=3.2 Hz). ¹³C NMR (100 MHz, CDCl₃) δ:205.2, 69.1, 52.4, 51.8, 49.1, 42.7, 40.5, 30.8, 34.3, 26.2, 25.8, 23.3,17.6, 14.1, 13.3, −4.7, −5.1.

Des-A,B-8β-[(tert-butyldimethylsilyl)oxy]-pregnan-20-one (6). A flamedried flask was charged with t-BuOK (1.55 g, 13.9 mmol) and anhydroust-BuOH (30 mL) at room temperature. O₂ was bubbled through the solutionfor 15 min. A solution of aldehyde 5 (0.9 g, 2.78 mmol) in anhydroust-BuOH (15 mL) was added to the reaction mixture and O₂ was bubbledthrough the solution for additional 10 min. The reaction was quenchedwith water (15 ml) and extracted with ether (3×30 mL). The combinedorganic phases were dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by column chromatography (3% ethyl acetate/hexane)to give 0.61 g (1.97 mmol, 71%) of the ketone 6. ¹H NMR (400 MHz, CDCl₃)δ: 0.01 (6H, s), 0.84 (3H, s), 0.87 (9H, s), 2.08 (3H, s), 2.46 (1H, t,J=9.1 Hz), 4.03 (1H, m). ¹³C NMR (100 MHz, CDCl₃) δ: 209.5, 69.0, 64.5,53.2, 43.7, 39.8, 34.2, 31.6, 25.8, 23.2, 21.8, 17.6, 15.5, −4.8, −5.2.

5-Bromo-2-methyl-2-pentanol (8). To a −20° C. cooled solution ofethyl-4-bromobutyrate 7 (5 g, 25.6 mmol) in anhydrous diethyl ether (50mL) was added 3M solution of methylmagnesium bromide in diethyl ether(17.1 mL, 6.11 g, 51.3 mmol) under argon atmosphere over a period of 30min. The reaction mixture was stirred at room temperature for overnight.Saturated ammonium chloride solution was added to hydrolyse the reactionmixture followed by 1N HCl solution to dissolve the inorganic saltsformed. The aqueous phase was extracted with ether (3×50 mL). Thecombined extracts were washed with water (100 mL), saturated NaClsolution (100 mL), dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by column chromatography to afford 3.1 g (17.1mmol, 67%) of tertiary alcohol 8. ¹H NMR (400 MHz, CDCl₃) δ: 1.27 (6H,s), 1.64 (2H, m), 1.96 (2H, m), 3.44 (2H, t, J=6.68 Hz).

5-Bromo-2methyl-2[(tert-butyldimethylsilyl)oxy]-pentane (9). To a −50°C. cooled solution of alcohol 8 (3 g, 16.6 mmol) in anhydrous CH₂Cl₂ (50mL) was added 2,6-lutidine (2.32 mL, 2.13 g, 19.89 mmol) followed byTBSOTf (4.57 mL, 5.26 g, 19.9 mmol). The solution was stirred at 0° C.for 15 min and water (10 mL) was added. The mixture was extracted withCH₂Cl₂ (3×40 mL), and combined organic phases were washed with INaqueous solution of NaOH (40 mL), dried (Na₂SO₄), filtered andconcentrated. The residue was purified by silica gel columnchromatography (5% ethyl acetate/hexane) to give 3.9 g (13.2 mmol, 80%)of 9. ^(H NMR ()400 MHz, CDCl₃) δ: 0.07 (6H, s), 0.85 (9H, s), 1.21 (6H,s), 1.55 (2H, m), 1.95 (2H, m), 3.41 (2H, t, J=6.8 Hz)

Des-A,B-cholest-20(21)-ene-8β,25-diol (16): A solution of5-bromo-2methyl-2[(tert-butyldimethylsilyl)oxy]pentane 9 (2.84 g, 9.68mmol) in anhydrous ether (20 mL, containing catalytic amount of iodine)was added dropwise to a stirred suspension of magnesium powder (0.23 g,9.68 mmol) in anhydrous diethyl ether (5 mL) at room temperature withoccasional warming it up to 35° C. under argon atmosphere. Afteraddition was complete the mixture was stirred for 1 hr at roomtemperature and for 1hr at 40° C. Then it was cooled to 0° C. and asolution of ketone 6 (0.6 g, 1.94 mmol) in anhydrous diethyl ether (10mL) was added dropwise over a period of 30 min. After stirring thereaction mixture at room temperature for 3h it was hydrolysed withaqueous solution of NH₄Cl (20 mL). The organic layer was separated andaqueous phase was extracted with ethyl acetate (3×30 mL). The combinedorganic phases were washed with water (40 mL), dried (Na₂SO₄) andevaporated. Column chromatography of the residue gave 0.95 g (94%) ofmixture of alcohols 10. Phosphorous oxychloride (3 mL) was addeddropwise to a solution of mixture of alcohols 10 (0.95 g, 1.8 mmol) inanhydrous pyridine (20 mL) under argon atmosphere. The reaction wasstirred at room temperature overnight and poured into ice-water andextracted with ether (3×20 mL). The organic layer was washed withsaturated CuSO₄ solution (30 mL), 1N HCl (30 mL), water (50 mL). Theorganic phase was dried (NaSO₄), filtered and concentrated. ColumnChromatography of crude mixture furnished 0.72 g (78%) of mixture ofolefins 11a, 11b, 12a, 12b, 13. The olefin mixture without furtherpurification was dissolved in methanol (20 mL) and p-toluenesulfonicacid monohydrate (p-TSA) (0.100 g) was added at 0° C. The reactionmixture was stirred at room temperature for 3 days [Additional amountsof p-TSA were successively added (100 mg, 24h; 75 mg, 36h; 50 mg, 48h)].Methanol was evaporated and residue was diluted with ethyl acetate (30mL). The organic phase was washed with saturated aqueous NaHCO₃ solution(20 mL) water (20 mL), dried (Na₂CO₃) and evaporated. The residue waspurified on column chromatography to yield 284 mg of mixture of olefinalcohols.

Des-A,B-20(21)-ene-8β,25-diol (16). The olefin alcohols were separatedon HPLC (9.4-mm×25-cm zorbax-sil column, 4 ml/min) using IPA/hexane(5/95) solvent system. Diol 17-20Z 15b and Diol 20-21 16 eluted outtogether at Rv=45 mL. The alcohols were oxidized together.

25-(Triethylsilyloxy)-des-A,B-cholest-20(21)-ene-8-one (22). To asolution of mixture of alcohols 15b and 16 (34 mg, 121 μmol) inanhydrous CH₂Cl₂ (5 mL) was added PDC (68 mg, 182 μmol) at rt. Afterstirring the reaction for 3 h under argon atmosphere the solution waspassed through a pad of celite with ethyl acetate. The filtrate wasconcentrated and applied on a Sep-Pak cartridge and eluted with ethylacetate/hexane (20/80) to give a mixture of ketones 30.2 mg (108 μmol,89%) as colourless oil. To a −50° C. cooled solution of ketones (30.2mg, 108 μmol) in anhydrous CH₂Cl₂ (10 mL) was added 2,6-lutidine (25 μL,23 mg, 217 μmol) followed by TESOTf (37 μL, 43 mg, 163 μmol). Thesolution was stirred at 0° C. for 15 min and water (10 mL) was added.The mixture was extracted with CH₂Cl₂ (3×5 mL), and combined organicphases were washed with 1N aqueous solution of NaOH (10 mL) dried(Na₂SO₄), filtered and concentrated. The residue was purified by HPLC(9.4-mm×25-cm Zorbax-Sil column, 4 ml/min) using ethyl acetate/hexane(5/95) solvent system. Pure ketone 22 8.7 mg (22 μmol, 20%) was elutedat R_(v)=38 mL as colorless oil. ¹H NMR (400.13 MHz, CDCl₃) δ: 0.56 (3H,s), 0.57 (6H, q, J=7.84 Hz), 0.94 (9H, t, J=7.96 Hz), 1.20 (6H, s), 4.83and 4.92 (1H and 1H, each s); ¹³CNMR (100 MHZ, CDCl₃) δ: 211.7, 148.1,110.7, 73.3, 61.6, 56.29, 50.3, 44.8, 40.8, 37.9, 37.8, 29.9, 25.5,24.0, 23.0, 18.9, 13.2, 7.1, 6.8.

MS m/z (relative intensity): No M⁺, 363 ([M−C₂H₅]⁺, 17), 334([M−2xC₂H₅]⁺, 4), 103 (100). Exact mass calculated for C₂₄H₄₄O₂SiNa[M+Na]⁺ is 415.3008, found 415.3004.

1α,25 Dihydroxy-20(21)-ene-2-methylene-19-norvitamin D₃ (24). To asolution of phosphine oxide 18 (62 mg, 106 μmol) in anhydrous THF (750μL) at −25° C. was slowly added PhLi 1.8 M in Di-n-butyl ether (65 μL,9.8 mg, 117 μmol) under argon with stirring. The solution turned deeporange. The mixture was stirred at that temperature for 20 min andcooled to −78° C. A precooled (−78° C.) solution of ketone 22 (8.7 mg,22 μmol) in anhydrous THF (100 μL) was added slowly. The mixture wasstirred under argon atmosphere at −78° C. for 3h and at 0° C. for 18h.Ethyl acetate was added and organic phase was washed with brine, dried(Na₂SO₄) and evaporated. The residue was applied on a Sep-Pak cartridge,and eluted with 1% ethyl acetate/hexane to give the 19-nor protectedvitamin derivative. The vitamin was further purified by HPLC(9.4-mm×25-cm Zorbax-Sil column, 4 ml/min) using hexane/IPA (99.95:0.05)solvent system. Pure compound 23, 14.8 mg (19 μmol, 88%) was eluted atR_(v)=18 mL as colourless oil.[α]²⁰ _(D)−9.74 (c 0.74, CHCl₃); UV (inhexane): λ_(max) 245.1, 253.8, 263.7 nm; ¹H NMR (400 MHz, CDCl₃) δ:0.034, 0.053, 0.076, 0.084 (each 3H, each s), 0.45 (3H, s), 0.56 (6H, q,J=7.86 Hz), 0.87, 0.90 (each 9H, each s), 0.95 (9H, t, J=7.88 Hz), 1.20(6H, s), 2.19 (1H, dd, J=12.4, 8.4 Hz), 2.35 (1H, dd, J=12.68, 1.88 Hz),2.47 (1H, dd, J=13.4, 4.96 Hz), 2.53 (1H, dd, J=13.2, 5.8 Hz), 2.83 (1H,dd, J=11.96, 2.16 Hz), 4.43 (2H, m), 4.81, 4.88, 4.93 and 4.97 (each 1H,each s), 5.86 and 6.22 (each 1H, each d, J=11 Hz); MS m/z (relativeintensity): No M⁺, 481(2), 263(100); Exact mass calculated forC₄₅H₈₄O₃Si₃Na [M+Na]⁺ is 779.5624, found 779.5629.

The protected vitamin 23 (14.8 mg, 19 mmol) was dissolved in anhydrousTHF (500 μL) and treated with TBAF (196 μL, 51.2 mg, 190 μmol) andstirred at rt in dark for overnight. The solvent was removed in vaccuoand residue was applied on Sep-Pak cartridge, and eluted with 30% ethylacetate/hexane to get the deprotected vitamin. The vitamin was furtherpurified by HPLC (9.4-mm×25-cm Zorbax-Sil column, 4 ml/min) usinghexane/IPA (85/15) as solvent system. Pure vitamin 24, 4.1 mg (9.9 μmol,51%) was eluted at R_(v)=33 mL. UV (in ethanol): λ_(max) 244.8, 253.2,262.8 nm; ¹H NMR (400 MHz, CDCl₃) δ: 0.46 (3H, s), 1.22 (6H, s), 1.57(3H, br s), 2.29 (2H, m), 2.57 (1H, dd, J=13.3, 3.6 Hz), 2.84 (2H, M),4.48 (2H, m), 4.83 and 4.88 (1H and 1H, each s), 5.09 and 5.11 (1H and1H, each s), 5.90 and 6.35 (1H and 1H, each d, J=11.2 Hz); MS m/z(relative intensity):414 (M⁺, 85), 396 (M−H₂O⁺, 23), 381 [M−CH₃—H₂O]⁺,10), 285 (20), 243 (30), 91 (100); exact mass calculated for C₂₇H₄₂O₃Na([M+Na]⁺) is 437.3032, measured is 437.3042.

Biological Activity of2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃

The introduction of a methylene group to the 2-position, and a doublebond between carbon atoms 20 and 21 in the side chain, had little effecton binding of Vit III 20-21 to the full length recombinant rat vitamin Dreceptor, as compared to 1α,25-dihydroxyvitamin D₃. The compound Vit III20-21 bound with the same affinity to the nuclear vitamin D receptor ascompared to the standard 1,25-(OH)₂D₃ (FIG. 1). It might be expectedfrom these results that compound Vit III 20-21 would have equivalentbiological activity. Surprisingly, however, compound Vit III 20-21 is ahighly selective analog with unique biological activity.

FIG. 5 shows that Vit III 20-21 has significant ability to increaseintestinal calcium transport activity in vivo, in a dose dependentmanner, and it clearly has about the same activity as compared to thatof 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the natural hormone, instimulating intestinal calcium transport. Vit III 20-21 stimulatedintestinal calcium transport as potently as 1,25(OH)₂D₃.

FIG. 4 demonstrates that Vit III 20-21 has less bone calciummobilization activity, as compared to 1,25(OH)₂D₃. Although Vit III20-21 has some bone calcium mobilization activity, it clearly has lowerpotency in mobilizing calcium from bone as compared to 1,25(OH)₂D₃.

FIGS. 4 and 5 thus illustrate that Vit III 20-21 may be characterized ashaving relatively significant intestinal calcium transport activity, butrelatively low bone calcium mobilization activity.

FIG. 2 illustrates that Vit III 20-21 is about 3 to 4 times more potentthan 1,25(OH)₂D₃ on HL-60 cell differentiation, i.e. causing thedifferentiation of HL-60 cells into monocytes, making it an excellentcandidate for the treatment of psoriasis and cancer, especially againstleukemia, colon cancer, breast cancer, skin cancer and prostate cancer.In addition, due to its relatively high cell differentiation activity,this compound provides a therapeutic agent for the treatment of variousskin conditions including wrinkles, lack of adequate dermal hydration,i.e. dry skin, lack of adequate skin firmness, i.e. slack skin, andinsufficient sebum secretion. Use of this compound thus not only resultsin moisturizing of skin but also improves the barrier function of skin.

FIG. 3 illustrates that in bone cells the compound Vit III 20-21 is onelog, i.e. 10 times, more potent than 1,25(OH)₂D₃ in increasingtranscription of the 24-hydroxylase gene. This result, together with thecell differentiation activity of FIG. 2, suggests that Vit III 20-21will be very effective in psoriasis because it has direct cellularactivity in causing cell differentiation, gene transcription, and insuppressing cell growth. These data also indicate that Vit III 20-21 mayhave significant activity as an anti-cancer agent, especially againstleukemia, colon cancer, breast cancer, skin cancer and prostate cancer.

The strong activity of Vit III 20-21 on HL-60 differentiation suggestsit will be active in suppressing growth of parathyroid glands and in thesuppression of the preproparathyroid gene.

Experimental Methods

Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3)Codon Plus RIL cells and purified to homogeneity using two differentcolumn chromatography systems. The first system was a nickel affinityresin that utilizes the C-terminal histidine tag on this protein. Theprotein that was eluted from this resin was further purified using ionexchange chromatography (S-Sepharose Fast Flow). Aliquots of thepurified protein were quick frozen in liquid nitrogen and stored at −80°C. until use. For use in binding assays, the protein was diluted inTEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCl) with 0.1%Chaps detergent. The receptor protein and ligand concentration wereoptimized such that no more than 20% of the added radiolabeled ligandwas bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient=18,200 and λ_(max)=265 nm; Analogs: molar extinctioncoefficient=42,000 and λ_(max)=252 nm). Radiolabeled ligand(³H-1,25(OH)₂D₃, ˜159 Ci/mmole) was added in ethanol at a finalconcentration of 1 nM.

Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of ≦10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After thefinal wash, the pellets were transferred to scintillation vialscontaining 4 ml of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (≦0.2%) present in the cell cultures.

Cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/ml. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

In vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably transfected with a 24-hydroxylase (24Ohase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.

RLU=relative luciferase units.

Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (0.47% Ca)diet+AEK oil for one week followed by Diet 11 (0.02% Ca)+AEK oil for 3weeks. The rats were then switched to a diet containing 0.47% Ca for oneweek followed by two weeks on a diet containing 0.02% Ca. Doseadministration began during the last week on 0.02% calcium diet. Fourconsecutive ip doses were given approximately 24 hours apart.Twenty-four hours after the last dose, blood was collected from thesevered neck and the concentration of serum calcium determined as ameasure of bone calcium mobilization. The first 10 cm of the intestinewas also collected for intestinal calcium transport analysis using theeverted gut sac method.

Interpretation of Data

Summary of Biological Findings. This compound binds the VDR with thesame affinity as the native hormone, and displays approximately 3 to 4times greater cell differentiation activity and more than 10 times invitro gene transcription activity compared to 1,25(OH)₂D₃. In vivo thiscompound exhibits significantly less bone calcium mobilization and aboutthe same intestinal calcium transport activities compared to the nativehormone making this compound a potentially valuable agent for thetreatment of such diseases as cancer, renal osteodystrophy, autoimmunediseases, skin conditions, and psoriasis. While this compound is morepotent compared to 1,25(OH)₂D₃ in vitro, it shows lower activity in vivoon bone calcium mobilization and similar activity in the intestinecompared to the native hormone. Vit III 20-21 remains a potentiallyvaluable compound for therapeutic development as it has lower potency inmobilizing calcium from bone storage, but higher potency in celldifferentiation potentially resulting in a compound with a bigger safetywindow than has previously been generated. Vit III 20-21 might not onlybe useful in the treatment of the above listed diseases, but also in theprevention of the above listed diseases.

VDR binding, HL60 cell differentiation, and transcription activity. VitIII 20-21 (K_(i)=1×10⁻¹⁰ M) is equally as active as the natural hormone1α,25-dihydroxyvitamin D₃ (K_(i)=5×10⁻¹¹ M) in its ability to competewith [³H]-1,25(OH)₂D₃ for binding to the full-length recombinant ratvitamin D receptor (FIG. 1). Vit III 20-21 displays about 3 to 4 timesgreater activity (EC₅₀=7×10⁻¹⁰ M) in its ability (efficacy or potency)to promote HL-60 cell differentiation as compared to1α,25-dihydroxyvitamin D₃ (EC₅₀=2×10⁻⁹ M) (See FIG. 2). Also, compoundVit III 20-21 (EC₅₀=2×10⁻⁹ M) has more than 10 times greatertranscriptional activity in bone cells than 1α,25-dihydroxyvitamin D₃(EC₅₀=3×10⁻¹⁰M) (See FIG. 3). These results suggest that Vit III 20-21will be very effective in psoriasis because it has direct cellularactivity in causing cell differentiation, gene transcription, and insuppressing cell growth. These data also indicate that Vit III 20-21will have significant activity as an anti-cancer agent, especiallyagainst leukemia, colon cancer, breast cancer, skin cancer and prostatecancer, as well as against skin conditions such as dry skin (lack ofdermal hydration), undue skin slackness (insufficient skin firmness),insufficient sebum secretion and wrinkles. It would also be expected tobe very active in suppressing secondary hyperparathyroidism.

Calcium mobilization from bone and intestinal calcium absorption invitamin D-deficient animals. Using vitamin D-deficient rats on a lowcalcium diet (0.02%), the activities of Vit III 20-21 and 1,25(OH)₂D₃ inintestine and bone were tested. As expected, the native hormone(1,25(OH)₂D₃) increased serum calcium levels at the 260 pmol/day dosage(FIG. 4). The study reported in FIG. 4 shows that Vit III 20-21 hasrelatively low or little activity in mobilizing calcium from bone.Administration of Vit III 20-21 at 260 pmol/day for 4 consecutive daysdid not result in mobilization of bone calcium, and it was not until theamount of Vit III 20-21 was increased to 2340 pmol/day that anysubstantial effect was seen.

Intestinal calcium transport was evaluated in the same groups of animalsusing the everted gut sac method (FIG. 5). These results show that thecompound Vit III 20-21 promotes intestinal calcium transport whenadministered at 260 pmol/day, and its activity is about the same orequal to 1,25(OH)₂D₃ which also provides a significant increase at the260 pmol/day dose. Thus, it may be concluded that Vit III 20-21 hassimilar intestinal calcium transport activity at the recommended lowerdoses to that of 1,25-(OH)₂D₃.

These results illustrate that Vit III 20-21 is an excellent candidatefor numerous human therapies as described herein, and that it may beparticularly useful in a number of circumstances such as suppression ofsecondary hyperparathyroidism of renal osteodystrophy, autoimmunediseases, cancer, numerous types of skin conditions, and psoriasis. VitIII 20-21 is an excellent candidate for treating psoriasis because: (1)it has significant VDR binding, transcription activity and cellulardifferentiation activity; (2) it has little hypercalcemic liability atrelatively low doses, unlike 1,25(OH)₂D₃; and (3) it is easilysynthesized. Since Vit III 20-21 has significant binding activity to thevitamin D receptor, but has little ability to raise blood serum calcium,it may also be particularly useful for the treatment of secondaryhyperparathyroidism of renal osteodystrophy.

These data also indicate that the compound Vit III 20-21 of theinvention may be especially suited for treatment and prophylaxis ofhuman disorders which are characterized by an imbalance in the immunesystem, e.g. in autoimmune diseases, including multiple sclerosis,lupus, diabetes mellitus, host versus graft rejection, and rejection oforgan transplants; and additionally for the treatment of inflammatorydiseases, such as rheumatoid arthritis, asthma, and inflammatory boweldiseases such as celiac disease, ulcerative colitis and Crohn's disease.Acne, alopecia and hypertension are other conditions which may betreated with the compound Vit III 20-21 of the invention.

The compounds of the invention of formula I, and particularly formulaIa, are also useful in preventing or treating obesity, inhibitingadipocyte differentiation, inhibiting SCD-1 gene transcription, and/orreducing body fat in animal subjects. Therefore, in some embodiments, amethod of preventing or treating obesity, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal subject includes administering to the animalsubject, an effective amount of one or more of the compounds or apharmaceutical composition that includes one or more of the compounds offormula I. Administration of the compound or the pharmaceuticalcompositions to the subject inhibits adipocyte differentiation, inhibitsgene transcription, and/or reduces body fat in the animal subject. Theanimal may be a human, a domestic animal such as a dog or a cat, or anagricultural animal, especially those that provide meat for humanconsumption, such as fowl like chickens, turkeys, pheasant or quail, aswell as bovine, ovine, caprine, or porcine animals.

For prevention and/or treatment purposes, the compounds of thisinvention defined by formula I, particulary Vit III 20-21, may beformulated for pharmaceutical applications as a solution in innocuoussolvents, or as an emulsion, suspension or dispersion in suitablesolvents or carriers, or as pills, tablets or capsules, together withsolid carriers, according to conventional methods known in the art. Anysuch formulations may also contain other pharmaceutically-acceptable andnon-toxic excipients such as stabilizers, anti-oxidants, binders,coloring agents or emulsifying or taste-modifying agents.

The compounds of formula I and particularly Vit III 20-21, may beadministered orally, topically, parenterally, rectally, nasally,sublingually or transdermally. The compound is advantageouslyadministered by injection or by intravenous infusion or suitable sterilesolutions, or in the form of liquid or solid doses via the alimentarycanal, or in the form of creams, ointments, patches, or similar vehiclessuitable for transdermal applications. A dose of from 0.01 μg to 1000 μgper day of the compounds I, particularly Vit III 20-21, preferably fromabout 0.1 μg to about 500 μg per day, is appropriate for preventionand/or treatment purposes, such dose being adjusted according to thedisease to be treated, its severity and the response of the subject asis well understood in the art. Since the compound exhibits specificityof action, each may be suitably administered alone, or together withgraded doses of another active vitamin D compound—e.g. 1α-hydroxyvitaminD₂ or D₃, or 1α,25-dihydroxyvitamin D₃—in situations where differentdegrees of bone mineral mobilization and calcium transport stimulationis found to be advantageous.

Compositions for use in the above-mentioned treatments comprise aneffective amount of the compounds I, particularly Vit III 20-21, asdefined by the above formula I and Ia as the active ingredient, and asuitable carrier. An effective amount of such compound for use inaccordance with this invention is from about 0.01 μg to about 1000 μgper gm of composition, preferably from about 0.1 μg to about 500 μg pergram of composition, and may be administered topically, transdermally,orally, rectally, nasally, sublingually, or parenterally in dosages offrom about 0.01 μg/day to about 1000 μg/day, and preferably from about0.1 μg/day to about 500 μg/day.

The compounds I, particularly Vit III 20-21, may be formulated ascreams, lotions, ointments, topical patches, pills, capsules or tablets,suppositories, aerosols, or in liquid form as solutions, emulsions,dispersions, or suspensions in pharmaceutically innocuous and acceptablesolvent or oils, and such preparations may contain in addition otherpharmaceutically innocuous or beneficial components, such asstabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compounds I, particularly Vit III 20-21, may be advantageouslyadministered in amounts sufficient to effect the differentiation ofpromyelocytes to normal macrophages. Dosages as described above aresuitable, it being understood that the amounts given are to be adjustedin accordance with the severity of the disease, and the condition andresponse of the subject as is well understood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For nasal administration, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

1. A compound having the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 2. The compound ofclaim 1 wherein X₃ is hydrogen.
 3. The compound of claim 1 wherein X₁ ishydrogen.
 4. The compound of claim 1 wherein X₁, X₂ and X₃ are allt-butyldimethylsilyl.
 5. A pharmaceutical composition containing aneffective amount of at least one compound as claimed in claim 1 togetherwith a pharmaceutically acceptable excipient.
 6. The pharmaceuticalcomposition of claim 5 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 7. Thepharmaceutical composition of claim 5 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 8.2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃ having theformula:


9. A pharmaceutical composition containing an effective amount of2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃ togetherwith a pharmaceutically acceptable excipient.
 10. The pharmaceuticalcomposition of claim 9 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 11. Thepharmaceutical composition of claim 9 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 12.A method of treating psoriasis comprising administering to a Subjectwith psoriasis an effective amount of a compound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 13. The method ofclaim 12 wherein the compound is administered orally.
 14. The method ofclaim 12 wherein the compound is administered parenterally.
 15. Themethod of claim 12 wherein the compound is administered transdermally.16. The method of claim 12 wherein the compound is administeredtopically.
 17. The method of claim 12 wherein the compound isadministered rectally.
 18. The method of claim 12 wherein the compoundis administered nasally.
 19. The method of claim 12 wherein the compoundis administered sublingually.
 20. The method of claim 12 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 21. The method of claim 12 wherein the compound is2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃ having theformula:


22. A method of treating a disease selected from the group consisting ofleukemia, colon cancer, breast cancer, skin cancer or prostate cancercomprising administering to a subject with said disease an effectiveamount of a compound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 23. The method ofclaim 22 wherein the compound is administered orally.
 24. The method ofclaim 22 wherein the compound is administered parenterally.
 25. Themethod of claim 22 wherein the compound is administered transdermally.26. The method of claim 22 wherein the compound is administeredrectally.
 27. The method of claim 22 wherein the compound isadministered nasally.
 28. The method of claim 22 wherein the compound isadministered sublingually.
 29. The method of claim 22 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 30. The method of claim 22 wherein the compound is2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃ having theformula:


31. A method of treating an autoimmune disease selected from the groupconsisting of multiple sclerosis, lupus, diabetes mellitus, host versusgraft rejection, and rejection of organ transplants, comprisingadministering to a subject with said disease an effective amount of acompound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 32. The method ofclaim 31 wherein the compound is administered orally.
 33. The method ofclaim 31 wherein the compound is administered parenterally.
 34. Themethod of claim 31 wherein the compound is administered transdermally.35. The method of claim 31 wherein the compound is administeredrectally.
 36. The method of claim 31 wherein the compound isadministered nasally.
 37. The method of claim 31 wherein the compound isadministered sublingually.
 38. The method of claim 31 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 39. The method of claim 31 wherein the compound is2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃ having theformula:


40. A method of treating an inflammatory disease selected from the groupconsisting of rheumatoid arthritis, asthma, and inflammatory boweldiseases, comprising administering to a subject with said disease aneffective amount of a compound having the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 41. The method ofclaim 40 wherein the compound is administered orally.
 42. The method ofclaim 40 wherein the compound is administered parenterally.
 43. Themethod of claim 40 wherein the compound is administered transdermally.44. The method of claim 40 wherein the compound is administeredrectally.
 45. The method of claim 40 wherein the compound isadministered nasally.
 46. The method of claim 40 wherein the compound isadministered sublingually.
 47. The method of claim 40 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 48. The method of claim 40 wherein the compound is2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃ having theformula:


49. A method of treating a skin condition selected from the groupconsisting of wrinkles, lack of adequate skin firmness, lack of adequatedermal hydration and insufficient sebum secretion which comprisesadministering to a subject with said skin condition an effective amountof a compound having the formula:

where X₁, X₂ and X₃ which may be the same or different, are eachselected from hydrogen or A hydroxy-protecting group.
 50. The method ofclaim 49 wherein the compound is administered orally.
 51. The method ofclaim 49 wherein the compound is administered parenterally.
 52. Themethod of claim 49 wherein the compound is administered transdermally.53. The method of claim 49 wherein the compound is administeredtopically.
 54. The method of claim 49 wherein the compound isadministered rectally.
 55. The method of claim 49 wherein the compoundis administered nasally.
 56. The method of claim 49 wherein the compoundis administered sublingually.
 57. The method of claim 49 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 58. The method of claim 49 wherein the compound is2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃ having theformula:


59. A method of treating renal osteodystrophy comprising administeringto a subject with renal osteodystrophy an effective amount of a compoundhaving the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 60. The method ofclaim 59 wherein the compound is administered orally.
 61. The method ofclaim 59 wherein the compound is administered parenterally.
 62. Themethod of claim 59 wherein the compound is administered transdermally.63. The method of claim 59 wherein the compound is administeredrectally.
 64. The method of claim 59 wherein the compound isadministered nasally.
 65. The method of claim 59 wherein the compound isadministered sublingually.
 66. The method of claim 59 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 67. The method of claim 59 wherein the compound is2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃ having theformula:


68. A method of treating obesity of an animal, inhibiting adipocytedifferentiation, inhibiting SCD-1 gene transcription, and/or reducingbody fat in an animal comprising administering to an animal in needthereof an effective amount of a compound having the formula:

where X₁, X₂ and X₃, which may be the same or different, are eachselected from hydrogen or a hydroxy-protecting group.
 69. The method ofclaim 68 wherein the compound is administered orally.
 70. The method ofclaim 68 wherein the compound is administered parenterally.
 71. Themethod of claim 68 wherein the compound is administered transdermally.72. The method of claim 68 wherein the compound is administeredrectally.
 73. The method of claim 68 wherein the compound isadministered nasally.
 74. The method of claim 68 wherein the compound isadministered sublingually.
 75. The method of claim 68 wherein thecompound is administered in a dosage of from about 0.01 μg/day to about1000 μg/day.
 76. The method of claim 68 wherein the compound is2-methylene-20(21)-dehydro-19-nor-1α,25-dihydroxyvitamin D₃ having theformula:


77. The method of claim 68 wherein the animal is a human.
 78. The methodof claim 68 wherein the animal is a domestic animal.
 79. The method ofclaim 68 wherein the animal is an agricultural animal.